Proteome Analysis of Orphan Plant Species, Fact or Fiction? - Plant Proteomics: Methods and Protocols - page 327

Fig. 3 Example from Fig. 2 , of Musa sucrose synthase. Nodes that represent a protein are visualized in white ,
peptides in red . Interactions with a ladder score between 0 and 50 are visualized in red , a score between 50 and
60 between in orange and >60 in green . Fifteen Musa specifi c EST sequences have at least one peptide with a
ladder score above 50. Those 15 EST sequences are all unique and have at least one amino acid difference. The
clustering compared to known full sequences of sucrose synthase shows that none of the Musa EST sequences
present in the database cover the whole protein range. The EST database is from a mixed origin (5-end and
3 -end; different varieties), which is illustrated in the clustering. Peptide nodes unique for one sequence have a

triangular shape. Homologous peptide nodes are colored in yellow . The Cytoscape view gives an impression
about the protein inference and shows that we have experimental evidence for at least two different isoforms.
Thirty percent of the peptides are not conserved and are unique for Musa (color fi gure online)

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4.3 Peptide
Separation, MS,
and Identifi cation

1. 2-D nanoAcquity UPLC system online coupled to a Synapt
HDMS Q-TOF MS instrument (Waters, Milford, MA, USA).
2. First RP (bridge C18, 5 μ m) column (300 μ m × 50 mm, Waters).

3. Second C18 (Symmetry 5 μ m) trap column (180 μ m × 20 mm,

Waters, Milford, MA, USA).
4. Syringe pump for lock spray.
5. Lock mass, [Glu1]fi brinopeptide B (1 pmol/L) (Waters,
Milford, MA, USA).
6. Proteinlynx global server (Waters, Milford, MA, USA).
7. Cytoscape http://www.cytoscape.org/ (free ware).
8. MS homology http://prospector.ucsf.edu/prospector/

mshome.htm
9. Microsoft Excel 2010.